The present invention relates generally to regulation of programmed cell death and more specifically to molecules that promote programmed cell death.
In essentially all self-renewing tissues, a balance is struck between cell production by mitogenesis and cell loss due to programmed cell death, thereby maintaining total cell numbers within a physiologically appropriate range. In pathological conditions, however, the balance in cell production and cell loss can be disrupted. In cancer, for example, an increased amount of cell production due to a shortened cell cycle time or a decreased amount of cell death due to dysregulation of a programmed cell death pathway results in the growth of a tumor.
With regard to programmed cell death, a variety of stimuli, which occur either external or internal to the cell, initiate a pathway that ultimately results in apoptosis of the cell. As is common for most signal transduction pathways, the various different stimuli that induce apoptosis likely initiate the process of programmed cell death through specific pathways. However, most if not all of these initial pathways converge at a common point that generally involves a member of the Bcl-2 family of proteins.
The Bcl-2 family of proteins regulate a distal step in the evolutionarily conserved pathway for programmed cell death and apoptosis, with some members of this family functioning as suppressors of cell death (anti-apoptotic proteins) and other members functioning as promoters of cell death (pro-apoptotic proteins). Overexpression of the anti-apoptotic protein, Bcl-2, for example, blocks neuronal cell death that otherwise is induced in vitro by various stimuli, including neurotrophic factor withdrawal, various oxidants, glucose deprivation, certain neurotrophic viruses, and amyloid β-peptide. In addition, Bcl-2 is overexpressed in some tumor cells and, in part, may contribute to tumor growth by altering the balance between cell division and cell death.
The Bcl-2 family of proteins are critical regulators of pathways involved in apoptosis, acting to either inhibit or promote cell death (Reed, Nature 387:773-776 (1997); Green and Reed, Science 281:1309-1312 (1998); Reed, Oncogene 17:3225-3236 (1998); Reed, Curr. Opin. Oncol. 11:68-75 (1999)). The Bcl-2 family members can be divided into two groups, those with anti-apoptotic activity, including Bcl-2 and Bcl-XL, and those with pro-apoptotic activity, including Bax and Bak.
Four distinct domains have been identified in Bcl-2 family members, designated BH1 to BH4. The BH4 domain is a domain that mediates interactions with a variety of cellular proteins (Reed, supra, 1998). The BH1, BH2 and BH3 domains form a binding pocket for dimerization with other Bcl-2 members having a BH3 domain, which also functions as a ligand that binds to the dimerization binding pocket. The dimerization function of the Bcl-2 members is an important mechanism for regulating apoptosis in that heterodimerization of pro-apoptotic Bcl-2 members with anti-apoptotic Bcl-2 members can regulate the cellular apoptotic pathways. Some Bcl-2 members only have a BH3 domain and therefore function as trans-dominant inhibitors of anti-apopototic proteins such as Bcl-2 and Bcl-XL (Reed, supra, 1998).
Another function of Bcl-2 members is the formation of ion channels. Bcl-2 members can localize to the mitochondrial membrane, and the formation of ion pores that alter the permeability of mitochondria is thought to be an important signaling mechanism for the induction of apoptosis. Thus, Bcl-2 members use at least three mechanisms to regulate apoptotic activity: dimerization with Bcl-2 members, formation of ion pores in mitochondria, and binding to non-Bcl-2 members that function as signaling molecules.
In comparison, overexpression of the pro-apoptotic protein, Bax, for example, promotes cell death when triggered by a variety of inducers of apoptosis, including growth factor withdrawal, ionizing radiation, and anti-Fas antibody. In addition, elevations in Bax expression occur in association with cell death induced by a variety of stimuli, including neuronal cell death that occurs due to ischemia, epilepsy, spinal cord injury, and certain neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.
Although aberrant expression of members of the Bcl-2 family of proteins is associated with various pathologic conditions, the mechanisms by which these proteins mediate their action is not known. Often, the action of a protein can be inferred from its structural relationship to other proteins, whose functions are known. However, while the Bcl-2 family proteins share certain structural homologies with each other, they do not share substantial amino acid sequence homology with other proteins, further hindering attempts to understand how the Bcl-2 family proteins such as Bcl-2 and Bax regulate cell death.
Thus, a need exists to identify proteins involved in the programmed cell death pathway and to identify methods of regulating programmed cell death for therapeutic applications, including treatment of cancer. The present invention satisfies this need and provides related advantages as well.